Any discussion of the background art throughout the specification should in no way be considered as an admission that such art is widely known or forms part of common general knowledge in the field.
Stem cells derived from the inner mass of mammalian blastocysts (embryonic stem cells, ESCs) have the ability to self-renew and differentiate into all body cell types (Evans and Kaufman, 1981). These properties have led to expectations that human embryonic stem cells (hESCs) might be useful to treat patients with various diseases and injuries, thereby revolutionizing regenerative medicine.
Cell transplantation therapy using stem cells may offer a viable treatment strategy for patients with disease or injury by (a) providing new cells to replace those lost through disease or injury, or (b) by replacing malfunctioning cells. However, the clinical application of hESCs faces difficulties regarding ethical concerns relating to the use of embryos, as well as instances of tissue rejection after implantation due to immunological incompatibility between patient and donor cells.
One way to circumvent these issues is to artificially derive an embryonic stem cell-like (pluripotent) cell from a mature somatic cell by inducing a “forced” expression of certain genes. These artificially derived embryonic stem cell-like cells are known as induced pluripotent stem (iPS) cells and are believed to be very similar to embryonic stem cells in many respects (Hochedlinger and Plath, 2009). The generation of iPS cells from mature somatic cells, such as fibroblast cells obtained directly from the patient, prevents therapeutic concerns regarding ethics, and may potentially provide the optimal cell source for regenerative medicine. Although immune rejection of autologous iPS cells (i.e. iPS cells derived from the patient) was not considered problematic, recent work suggests that immune rejection of autologous iPS cells may occur (Lister et al., 2011). At the same time, the tumorigenic properties of iPS cells (Ghosh et al., 2011) may prohibit their usage in clinical applications. Moreover, more recent work indicates that iPS cells appear to retain a memory of their somatic program (Ohi et al., 2011) and are characterised by genomic instability (Bar-Nur et al., 2011; Pasi et al., 2011)—another hurdle for the clinical application of such cells.
iPS cells were first generated by Yamanaka and colleagues in 2006 from mouse fibroblast cells (Cell 126, 663-676). The method of deriving iPS cells traditionally involves the transfection of certain embryonic stem cell-associated genes into non-pluripotent cells, such as mature fibroblasts. Transfection is usually achieved through viral vectors, such as retroviruses. Yamanaka and colleagues initially identified 4 key genes essential for the production of pluripotent stem cells: Oct-3/4, Sox2, c-Myc and Klf4. Additional studies demonstrated the requirement of Nanog as another major determinant of cellular pluripotency (Okita et al., 2007; Wernig et al., 2007; and Maherali et al., 2007). In 2007, two independent research groups generated iPS cells from human cells (Takahashi et al., 2007; and Yu et al., 2007). Applying the same principles used earlier in mouse cells, Yamanaka and colleagues (Takahashi et al., 2007) successfully transformed human fibroblasts into pluripotent stem cells using the same 4 pivotal genes Oct-3/4, Sox2, c-Myc and Klf4 in a retroviral transfection system. Thomson and colleagues (Yu et al., 2007) used Oct4, Sox2, Nanog and Lin28 using a lentiviral transfection system. The exclusion of c-Myc in these experiments was based on evidence that c-Myc is oncogenic and is not necessary to promote cellular pluripotency.
Alternatively, and as self-renewing stem cells also exist in adult tissues buffered within specialised niches, a further approach would be to identify a pluripotent stem cell population which can conveniently be harvested without detriment to a donor. Stem cells have been isolated from a wide range of adult tissues, from those that have a high rate of ongoing cellular turnover, such as blood, cord blood, bone marrow, skin, intestine, and breast tissue, to those with a low turnover such as brain, skeletal muscle, and juvenile teeth.
However, most adult self-renewing stem cells only escape their quiescent state in response to developmental and physical cues in order to maintain homeostasis during tissue turnover or injury (Bjornson et al., 1999) and, as such, are generally only found in populations of very limited numbers. Further, it was thought that adult stem cells have limited differentiation potential restricted to the tissue/organ of origin. However, recent evidence has suggested that certain cell subpopulations within adult stem cell compartments are capable of in vitro and in vivo differentiation into cell types unrelated to their developmental origin (Clarke et al., 2000; Krause et al., 2001; Jiang et al., 2002; Hoffman, 2007). Notwithstanding, so far only a few such cell populations have been identified and the pluripotent cells obtained are often very limited in number rarely allowing direct therapeutic application of the cells. Great research efforts are now being directed at establishing methods of expansion of such cell populations without compromising the cells' pluripotent properties and therefore their differentiation potential.
In light of the above, it is noteworthy that bi-potent stem cells (Mammary gland stem cells; MaSCs) have been identified in the mammary gland and are known to exist in the basal epithelial layer in the resting breast. Interestingly, these cells have been shown to be capable of differentiation, however, only towards the two main mammary cell lineages, the myoepithelial and luminal cell lineages (Dontu et al., 2003; Shackleton et al., 2006; Villadsen et al., 2007; Visvader, 2009).
The mammary gland undergoes significant remodelling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSCs) proliferation. Briefly, while rudimentary mammary glands exist in males and females, hormonal changes during puberty lead to further development in females mainly through the influence of oestrogen and progesterone leading to the establishment of a rudimentary duct structure with terminal end bud formation. While, influenced by progesterone and prolactin levels, the duct structure of the mammary gland is further refined following puberty, secretory alveoli only develop during pregnancy. The gland finally develops into a secretory, milk producing organ during late pregnancy and/or in the first few days after birth with the secretion of colostrum. Colostrum production is followed by a short period of production of the so called “transition milk” leading to the production of mature milk throughout the lactation period. After weaning, the mammary gland undergoes involution during which the developmental stages seen during gestation are reversed such that the mammary gland reverts to a near resting state. From the above, it will be appreciated that the mammary gland is a highly plastic organ capable of intense remodelling during repeated pregnancies during the life of a female.
The lactating breast in its fully mature state displays the complete cellular hierarchy and contains a larger number of stem cells than found in the resting breast. It is therefore thought that the lactating breast provides the best model for studying the properties of MaSCs. However, due to the scarcity of human lactating breast tissue specimens the human lactating breast has not been extensively studied in this context. As such, the scarcity of human lactating breast tissue specimens and the low number and quiescent state of MaSCs in available resting breast tissue specimens have hindered understanding of the normal MaSC biology and the molecular determinants that drive their aberrant self-renewal in breast cancer.
The existence of stem cells in the breast was first postulated based on the capacity of the mammary epithelium to significantly expand and regress in a repeated fashion throughout adult life (Taylor-Papadimitriou et al., 1977). In accordance with this hypothesis, and as known for other adult stem cell populations, MaSCs exist in a quiescent state and in scarce numbers in the resting breast. Yet, due to the physiological changes linked to mammary remodelling during pregnancy and lactation, MaSCs appear to undergo a controlled program of proliferation, differentiation, and apoptosis stimulated by hormonally-driven cues (Pang and Hartmann, 2007; Asselin-Labat et al., 2010; Joshi et al., 2010).
However, in addition to their important physiological role during pregnancy and lactation, increasing evidence also implicates MaSCs in the onset and progression of invasive breast carcinomas having metastatic features with a propensity towards both mesodermal (bone) and endodermal (lung) organs. It would therefore appear that the mammary gland harbours a stem cell population with unique self-renewal capabilities and a potential plasticity reflected both in normal development and in aberrant conditions of the breast. To date, and despite ongoing research in this field, our understanding of normal MaSC biology is limited and the molecular determinants and regulators of MaSC self-renewal and plasticity (normal or aberrant) are largely unknown.
Currently a lack of suitable cell culture model systems for propagation and characterisation of MaSCs exists and progress has been hindered twofold, (a) by the scarcity and quiescent state of MaSCs obtainable from the resting breast, and (b) by the extremely limited availability of human lactating breast tissue, which contains activated MaSCs in greater numbers.
Interestingly, it has also been shown that breastmilk contains a mixture of different cell types such as, for example, epithelial cells (lactocytes). It would appear that these cells are sloughing off the basement membrane of the breast as a consequence of the pressure and shear forces associated with the continued filling and emptying of the breast and, as a result infiltrate the milk. It has been stipulated that lactocytes account for approximately 10-20% of the total cell population (varying significantly between individual breastmilk samples). While it has long been thought that the majority of the remainder of cells found in human milk are immune cells (such as lymphocytes, macrophages, monocytes, natural killer cells, basophils, eosinophils, and neutrophils) more recent data suggests that human breastmilk from healthy subjects only contains a small percentage of immune cells.
In addition to lactocytes and immune cells, it has previously been demonstrated that breastmilk also contains an activated MaSC population derived from the lactating breast and that this cell population can be accessed non-invasively via expressed breastmilk (Cregan et al., 2007; Thomas et al., Cell Cycle, 2011; Thomas et al., Cell Death Dis., 2011). MaSCs as well as the differentiated cells from the lactating epithelium mentioned above enter breastmilk either through cell migration and turnover and/or as a consequence of the mechanical shear forces of breastfeeding. Culture of CD49f+, human breastmilk-derived multipotent cells expressing the epithelial stem cell marker p63 in epithelial-specific two-dimensional (2D) monolayer culture over a twenty-one day period showed that a non-adherent, multipotent p63+ epithelial cell population expands in the floating cell population within the first five days of growth. However, these cells are depleted reciprocally with attachment and expansion of adherent cells homogenously expressing pan-epithelial markers. In the proceeding sixteen days of growth subpopulations of adherent cells expressing myoepithelial and luminal epithelial markers in mutually exclusive colonies have been identified, indicating that lineage commitment has already occurred in the floating cell population prior to adherence. Furthermore, when the adherent cells are subsequently cultivated in a three-dimensional (3D) biomatrix, these cells have been shown to form differentiated spherical, milk protein-secreting structures (mammospheres) resembling the in vivo secretory alveoli. Based on these results, it would appear that the p63+/CD49f+ cell population in breastmilk shares properties of MaSCs (Thomas et al., Cell Cycle, 2011; Thomas et al., Cell Death Dis., 2011). As such, these results indicate that breastmilk contains MaSCs capable of differentiation towards the myoepithelial and luminal mammary lineages.
As indicated above, it has long been known that breastmilk contains a mixture of different cell types. However, a comprehensive analysis and characterisation of human milk identifying all different cell types found in breastmilk is still needed.
In light of the above, it will be appreciated that there is a need in the art for improved methods of isolating embryonic stem cell-like (pluripotent) cell populations from adult tissues/organs as well as for improved methods of propagation and expansion of any such cells with a view to producing cell numbers suitable for therapeutic application.